The present invention is directed to a DNA construct to confer multiple traits on plants.
Control of plant virus diseases took a major step forward in the last decade when it was shown in 1986 that the tobacco mosaic virus (xe2x80x9cTMVxe2x80x9d) coat protein gene that was expressed in transgenic tobacco conferred resistance to TMV (Powell-Abel, P., et al., xe2x80x9cDelay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene,xe2x80x9d Science, 232:738-43 (1986)). The concept of pathogen-derived resistance (xe2x80x9cPDRxe2x80x9d), which states that pathogen genes that are expressed in transgenic plants will confer resistance to infection by the homologous or related pathogens (Sanford, J. C., et al. xe2x80x9cThe Concept of Parasite-Derived Resistancexe2x80x94Deriving Resistance Genes from the Parasite""s Own Genome,xe2x80x9d J. Theor. Biol., 113:395-405 (1985)) was introduced at about the same time. Since then, numerous reports have confirmed that PDR is a useful strategy for developing transgenic plants that are resistant to many different viruses (Lomonossoff, G. P., xe2x80x9cPathogen-Derived Resistance to Plant Viruses,xe2x80x9d Ann. Rev. Photopathol., 33:323-43 (1995)).
Only eight years after the report by Beachy and colleagues (Powell-Abel, P., et al., xe2x80x9cDelay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene,xe2x80x9d Science, 232:738-43 (1986)), Grumet, R., xe2x80x9cDevelopment of Virus Resistant Plants via Genetic Engineering,xe2x80x9d Plant Breeding Reviews, 12:47-49 (1994) reviewed the PDR literature and listed the successful development of virus resistant transgenic plants to at least 11 different groups of plant viruses. The vast majority of reports have utilized the coat protein genes of the viruses that are targeted for control. Although the testing of transgenic plants have been largely confined to laboratory and greenhouse experiments, a growing number of reports showed that resistance is effective under field conditions (e.g., Grumet, R., xe2x80x9cDevelopment of Virus Resistant Plants via Genetic Engineering,xe2x80x9d Plant Breeding Reviews, 12:47-49 (1994)). Two virus resistant crops have been deregulated by APHIS/USDA and thus are approved for unrestricted release into the environment in the U.S.A. Squash that are resistant to watermelon mosaic virus 2 and zucchini yellow mosaic potyviruses have been commercialized (Fuchs, M., et al., xe2x80x9cResistance of Transgenic Hybrid Squash ZW-20 Expressing the Coat Protein Genes of Zucchini Yellow Mosaic Virus and Watermelon Mosaic Virus 2 to Mixed Infections by Both Potyviruses,xe2x80x9d Bio/Technoloqy, 13:1466-73 (1995); Tricoli, D. M., et al., xe2x80x9cField Evaluation of Transgenic Squash Containing Single or Multiple Virus Coat Protein Gene Constructs for Resistance to Cucumber Mosaic Virus, Watermelon Mosaic Virus 2, and Zucchini Yellow Mosaic Virus,xe2x80x9d Bio/Technology, 13:1458-65 (1995)). Also, a transgenic papaya that is resistant to papaya ringspot virus has been developed (Fitch, M. M. M., et al., xe2x80x9cVirus Resistant Papaya Derived from Tissues Bombarded with the Coat Protein Gene of Papaya Ringspot Virus,xe2x80x9d Bio/Technology, 10:1466-72 (1992); Tennant, P. F., et al., xe2x80x9cDifferential Protection Against Papaya Ringspot Virus Isolates in Coat Protein Gene Transgenic Papaya and Classically Cross-Protected Papaya,xe2x80x9d Phytopathology, 84:1359-66 (1994)). This resistant transgenic papaya was recently deregulated by USDA/APHIS. Deregulation of the transgenic papaya is timely, because Hawaii""s papaya industry is being devastated by papaya ringspot virus. Undoubtedly, more crops will be deregulated and commercialized in the near future.
Interestingly, remarkable progress has been made in developing virus resistant transgenic plants despite a poor understanding of the mechanisms involved in the various forms of pathogen-derived resistance (Lomonossoff, G. P., xe2x80x9cPathogen-Derived Resistance to Plant Viruses,xe2x80x9d Ann. Rev. Photopathol., 33:323-43 (1995)). Although most reports deal with the use of coat protein genes to confer resistance, a growing number of reports have shown that viral replicase (Golemboski, D. B., et al., xe2x80x9cPlants Transformed with a Tobacco Mosaic Virus Nonstructural Gene Sequence are Resistant to the Virus,xe2x80x9d Proc. Natl. Acad. Sci. USA, 87:6311-15 (1990)), movement protein (e.g., Beck, D. L., et al., xe2x80x9cDisruption of Virus Movement Confers Broad-Spectrum Resistance Against Systemic Infection by Plant Viruses with a Triple Gene Block,xe2x80x9d Proc. Natl. Acad. Sci. USA, 91:10310-14 (1994)), NIa proteases of potyviruses (e.g., Maiti, I. B., et al., xe2x80x9cPlants that Express a Potyvirus Proteinase Gene are Resistant to Virus Infection,xe2x80x9d Proc. Natl. Acad. Sci. USA, 90:6110-14 (1993)), and other viral genes are effective. This led to the conclusion that any part of a plant viral genome gives rise to PDR. Furthermore, the viral genes can be effective in the translatable and nontranslatable sense forms, and less frequently antisense forms (e.g., Baulcombe, D.C., xe2x80x9cMechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants,xe2x80x9d Plant Cell, 8:1833-44 (1996); Dougherty, W. G., et al., xe2x80x9cTransgenes and Gene Suppression: Telling us Something New?,xe2x80x9d Current Opinion in Cell Biology, 7:399-05 (1995); Lomonossoff, G. P., xe2x80x9cPathogen-Derived Resistance to Plant Viruses,xe2x80x9d Ann. Rev. Photopathol., 33:323-43 (1995)).
RNA-mediated resistance is the form of PDR where there is clear evidence that viral proteins do not play a role in conferring resistance to the transgenic plant. The first clear cases for RNA-mediated resistance were reported in 1992 for tobacco etch (xe2x80x9cTEVxe2x80x9d) potyvirus (Lindbo, et al., xe2x80x9cPathogen-Derived Resistance to a Potyvirus Immune and Resistance Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,xe2x80x9d Mol. Plant Microbe Interact., 5:144-53 (1992)), for potato virus Y (xe2x80x9cPVYxe2x80x9d) potyvirus by Van Der Vlugt, R. A. A., et al., xe2x80x9cEvidence for Sense RNA-Mediated Protection to PVY in Tobacco Plants Transformed with the Viral Oat Protein Cistron,xe2x80x9d Plant Mol. Biol., 20:631-39 (1992), and for tomato spotted wilt (xe2x80x9cTSWVxe2x80x9d) tospovirus by de Haan, P., et al., xe2x80x9cCharacterization of RNA-Mediated Resistance to Tomato Spotted Wilt Virus in Transgenic Tobacco Plants,xe2x80x9d Bio/Technology, 10:1133-37 (1992). Others confirmed the occurrence of RNA-mediated resistance with potyviruses (Smith, H. A., et al., xe2x80x9cTransgenic Plant Virus Resistance Mediated by Untranslatable Sense RNAs: Expression, Regulation, and Fate of Nonessential RNAs,xe2x80x9d Plant Cell, 6:1441-53 (1994)), potexviruses (Mueller, E., et al., xe2x80x9cHomology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,xe2x80x9d Plant Journal, 7:1001-13 (1995)), and TSWV and other topsoviruses (Pang, S. Z., et al., xe2x80x9cResistance of Transgenic Nicotiana Benthamiana Plants to Tomato Spotted Wilt and Impatiens Necrotic Spot Tospoviruses: Evidence of Involvement of the N Protein and N Gene RNA in Resistance,xe2x80x9d Phytopathology, 84:243-49 (1994); Pang, S.-Z., et al., xe2x80x9cDifferent Mechanisms Protect Transgenic Tobacco Against Tomato Spotted Wilt Virus and Impatiens Necrotic Spot Tospoviruses,xe2x80x9d Bio/Technology 11:819-24 (1993)). More recent work has shown that RNA-mediated resistance also occurs with the comovirus cowpea mosaic virus (Sijen, T., et al., xe2x80x9cRNA-Mediated Virus Resistance: Role of Repeated Transgene and Delineation of Targeted Regions,xe2x80x9d Plant Cell, 8:2227-94 (1996)) and squash mosaic virus (Jan, F.-J., et al., xe2x80x9cGenetic and Molecular Analysis of Squash Plants Transformed with Coat Protein Genes of Squash Mosaic Virus,xe2x80x9d Phytopathology, 86:S16-17 (1996)).
Major advances towards understanding the mechanism(s) of RNA-mediated resistance were made by Dougherty and colleagues in a series of experiments with TEV and PVY. Using TEV, Lindbo, J. A., xe2x80x9cPathogen-Derived Resistance to a Potyvirus Immune and Resistant Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,xe2x80x9d Mol. Plant Microbe Interact., 5:144-53 (1992) and Lindbo, J. A., et al., xe2x80x9cUntranslatable Transcripts of the Tobacco Etch Virus Coat Protein Gene Sequence can Interfere with Tobacco Etch Virus Replication in Transgenic Plants and Protoplasts,xe2x80x9d Virology, 189:725-33 (1992) showed that transgenic plants expressing translatable full length coat protein, truncated translatable coat protein, antisense coat protein genes, and nontranslatable coat protein gene had various phenotypic reactions after inoculation with TEV. Transgenic plants displayed resistance, recovery (inoculated plants initially show systemic infection but younger leaves that develop later are symptomless and resistant to the virus), or susceptible phenotypes. Furthermore, they showed that leaves of resistant plants and asymptomatic leaves of recovered plants had relatively low levels of steady state RNA when compared to those in leaves of susceptible plants (Lindbo, J. A., et al., xe2x80x9cInduction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance,xe2x80x9d Plant Cell, 5:1749-59 (1993)). However, nuclear run off experiments showed that those plants with low levels of steady state RNA had higher transcription rates of the viral transgene than those plants that were susceptible (and had high steady state RNA levels). To account for these observations, it was proposed xe2x80x9cthat the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivationxe2x80x9d (Lindbo, J. A., et al., xe2x80x9cInduction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance,xe2x80x9d Plant Cell, 5:1749-59 (1993)). It was also suggested that the low steady state RNA levels may be due to post-transcriptional gene silencing, a phenomenon that was first proposed by de Carvalho, F., et al., xe2x80x9cSuppression of beta-1,3-glucanase Transgene Expression in Homozygous Plants,xe2x80x9d EMBO J., 11:2595-602 (1992) for the suppression of xcex2-1,3-glucanase transgene in homozygous transgenic plants.
An RNA threshold model was proposed to account for the observations (Lindbo, J. A., et al., xe2x80x9cInduction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance,xe2x80x9d Plant Cell, 5:1749-59 (1993)). Basically, the model states that there is a cytoplasmic cellular degradation mechanism that acts to limit the RNA levels in plant cells, and that this mechanism is activated when the transgenic RNA transcript goes above a threshold level. The degradation mechanism is specific for the transcript that goes above the threshold level; and if the transcripts that go above a certain threshold is a viral transgene, the virus resistance state is observed in the plant, because the degradation mechanism also targets, for inactivation, the specific sequences of the incoming virus. The model also accounts for the xe2x80x98recoveryxe2x80x99 of transgenic plants by suggesting that viral RNA from the systemically invading virus triggers the phenomenon in some transgenic plants that have two copies of the transgenes. Plants that had more than three copies of the transgenes caused the threshold level to be surpassed without the invasion of virus (Goodwin, J., et al., xe2x80x9cGenetic and Biochemical Dissection of Transgenic RNA-Mediated Virus Resistance,xe2x80x9d Plant Cell, 8:95-105 (1996); Smith, H. A., et al., xe2x80x9cTransgenic Plant Virus Resistance Mediated by Untranslatable Sense RNAs: Expression, Regulation, and Fate of Nonessential RNAs,xe2x80x9d Plant Cell, 6:1441-53 (1994)). Although the degradation mechanism is not clear, it is proposed that a cellular RNA dependent RNA polymerase (xe2x80x9cRdRpxe2x80x9d) binds to the transcript and produces small fragments of antisense RNA which then bind to other transcripts to form duplexes which are then degraded by nucleases that specifically recognize RNA-RNA duplexes. This degradation mechanism is sequence specific, which accounts for the specificity of RNA mediated resistance.
Work on PVX by Baulcombe and colleagues (English, J. J., et al., xe2x80x9cSuppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes,xe2x80x9d Plant Cell, 8: 179-88 (1996); Mueller, E., et al., xe2x80x9cHomology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,xe2x80x9d Plant Journal, 7:1001-13 (1995)) confirmed and extended the results by Dougherty and colleagues. An aberrant RNA model which is a modification of the RNA threshold model proposed by Dougherty and colleagues was proposed. The features of the model are similar to Dougherty""s except that it states that the RNA level is not the sole trigger to activate the cellular degradation mechanism, but instead aberrant RNA that are produced during the transcription of the transgene plays an important part in activating the cytoplasmic cellular mechanism that degrades specific RNA. The production of aberrant RNA may be enhanced by positional affects of the transgene on the chromosome and by methylation of the transgene DNA. The precise nature of the aberrant RNA is not defined, but it may contain a characteristic that makes it a preferred template for the at production of antisense RNA by the host encoded RdRp (Baulcombe, D. C., xe2x80x9cMechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants,xe2x80x9d Plant Cell, 8:1833-44 (1996); English, J. J., et al., xe2x80x9cSuppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes,xe2x80x9d Plant Cell, 8: 179-88 (1996)). Thus, the model also proposes that RdRp and antisense molecules are involved in the degradation mechanism. Baulcombe and colleagues confirmed that plants which show low steady state transgene levels have multiple copies of transgenes and that the low steady state RNA and the accompanying resistant state is due to post-transcriptional gene silencing. The term homology-dependent resistance was proposed to describe the resistance in plants that show homology-dependent gene silencing (Mueller, E., et al., xe2x80x9cHomology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,xe2x80x9d Plant Journal, 7:1001-13 (1995)).
Experiments with TSWV tospovirus (Pang, S. Z., et al., xe2x80x9cPost-Transcriptional Transgene Silencing and Consequent Tospovirus Resistance in Transgenic Lettuce are Affected by Transgene Dosage and Plant Development,xe2x80x9d Plant Journal, 9:899-09 (1996); Prins, M., et al., xe2x80x9cEngineered RNA Mediated Resistance to Tomato Spotted Wilt Virus is Sequence Specific,xe2x80x9d Mol. Plant Microbe Interact., 9:416-18 (1996)) and cowpea mosaic comovirus (Sijen, T., et al., xe2x80x9cRNA-Mediated Virus Resistance: Role of Repeated Transgene and Delineation of Targeted Regions,xe2x80x9d Plant Cell, 8:2227-94 (1996)) also showed that resistance in transgenic plants is a consequence of post-transcriptional gene silencing. Pang, S. Z., et al., xe2x80x9cPost-Transcriptional Transgene Silencing and Consequent Tospovirus Resistance in Transgenic Lettuce are Affected by Transgene Dosage and Plant Development,xe2x80x9d Plant Journal, 9:899-09 (1996) showed that post-transcriptional gene silencing in transgenic lettuce expressing the N gene of TSWV was influenced by gene dosage and by the developmental stage of the plant. The effect of developmental stage on post-transcriptional gene silencing of transgenes and their effect on resistance had not been previously shown for transgenic plants expressing viral genes, but had been shown to occur in plants expressing other transgenes (de Carvalho, F., et al., xe2x80x9cSuppression of beta-1,3-glucanase Transgene Expression in Homozygous Plants,xe2x80x9d EMBO J., 11:2595-02 (1992)). Post-transcriptional gene silencing could also account for the correlation of low steady state level of N gene RNA in transgenic tobacco showing very high but specific resistance (Pang, S. Z., et al., xe2x80x9cDifferent Mechanisms Protect Transgenic Tobacco Against Tomato Spotted Wilt and Impatiens Necrotic Spot Tospoviruses,xe2x80x9d Bio/Technology, 11:819-24 (1993)). Prins, M., et al., xe2x80x9cEngineered RNA-Mediated Resistance to Tomato Spotted Wilt Virus is Sequence Specific,xe2x80x9d Molecular Plant Microbe Interactions, 9:416-18 (1996) also reported that post-transcriptional gene silencing occurred with transgenic tobacco expressing the N gene and nonstructural gene of the mRNA. Interestingly, it was found that tobacco with other parts of the TSWV genome were not resistant. They suggested, as one explanation, that those gene fragments which did not confer resistance may not fit the criteria for inducing post-transcriptional gene silencing. Sijen, T., et al., xe2x80x9cRNA-Mediated Virus Resistance: Role of Repeated Transgene and Delineation of Targeted Regions,xe2x80x9d Plant Cell, 8:2227-94 (1996) showed that resistance of transgenic plants expressing the movement protein, replicase, or coat protein were due to post-transcriptional gene silencing. This data also suggested that the 3xe2x80x2 region of the movement protein transgene mRNA is the initial target of the silencing mechanism.
The present invention is directed to producing improved disease resistant plants.
The present invention is directed to a DNA construct formed from a fusion gene which includes a trait DNA molecule and a silencer DNA molecule. The trait DNA molecule has a length that is insufficient to impart a desired trait to plants transformed with the trait DNA molecule. The silencer DNA molecule is operatively coupled to the trait DNA molecule with the trait and silencer DNA molecules collectively having sufficient length to impart the trait to plants transformed with the DNA construct. Expression systems, host cells, plants, and plant seeds containing the DNA construct are disclosed.
In an alternative embodiment of the present invention, the DNA construct can be a fusion gene comprising a plurality of trait DNA molecules at least some of which having a length that is insufficient to impart that trait to plants transformed with that trait DNA molecule. However, the plurality of trait DNA molecules collectively have a length sufficient to impart their traits to plants transformed with the DNA construct and to effect post-transcriptional silencing of the fusion gene. Expression systems, host cells, plants, and plant seeds containing this embodiment of the DNA construct are disclosed.
The present invention is particular directed to preparing plants which are resistant to multiple viruses. It is well known that particular plant types are often susceptible to more than one virus. Although PDR is an excellent approach to controlling the damaging effects of plant viruses, incorporating multiple virus resistance in a given plant can be challenging. For example, identifying and producing full length viral genes to transform plants can be expensive and time consuming. Further, such genes may be so large that they need to be incorporated in different expression systems which must be separately incorporated in plants.
Rather than attempting to incorporate full length viral genes in a plant, the present invention uses short fragments of such genes to impart resistance to the plant against a plurality of viral pathogens. These short fragments, which each by themselves have insufficient length to impart such resistance, are more easily and cost effectively produced than full length genes. There is no need to include in the plant separate promoters for each of the fragments; only a single promoter is required. Moreover, such viral gene fragments can preferably be incorporated in a single expression system to produce transgenic plants with a single transformation event.
The impact of this simple strategy for multiple virus resistant transgenic plants could have far reaching effects in agriculture. An example is the case of papaya ringspot virus (xe2x80x9cPRVxe2x80x9d). Transgenic papaya with the coat protein gene of the PRV strain from Hawaii have been developed and found to be highly resistant under greenhouse and long term field conditions. However, that papaya is largely susceptible to strains from other parts of the world, including Jamaica, Thailand, and Brazil. Apparently, PRV resistance in papaya is highly specific and a number of transgenic papaya lines will need to be developed with different coat protein genes of the target countries to control the virus worldwide. With the present invention, a transgenic papaya could be developed with resistance to all PRV strains using viral gene fragments that total less than 1,000 base pairs plus a silencer DNA of about 400 bp; by comparison, the PRV coat protein gene alone is about 1,000 bp.
Another use of the present invention involves imparting resistance against a plurality of different viruses. For example, in potato, the present invention can be employed to impart resistance against potato leaf roll virus, potato virus Y, and potato virus X. To effect such resistance, in accordance with the present invention, a DNA construct, driven by a single promoter, and containing a portion of the potato leafroll virus replicase encoding gene, a portion of the potato virus Y coat protein encoding gene, and a portion of the gene encoding the movement protein of potato virus X can be produced and transformed into potato. As a result, transgenic potato with resistance to potato leafroll virus, potato virus Y, and potato virus X can be produced by a single transformation event. This constitutes a significant advance beyond incorporating full length versions of each of the genes with separate promoters together in a single expression vector or in separate vectors.
Another use of the present invention involves imparting resistance to cucurbits against a number of viruses. For example, in squash, the present invention can be utilized to impart multiple resistance to zuccinni yellow mosaic virus, papaya ringspot virus, watermelon mosaic virus II, and squash mosaic virus. For example, a construct containing a portion of the coat protein encoding gene or a portion of the replicase encoding gene from each of these viruses, driven by a single promoter, can be produced and transformed into squash. The resulting transgenic squash is resistant to all of these viruses.
In addition to conferring on plants resistance to multiple viral diseases, the present invention can be utilized to impart other traits to plants. It is often desirable to incorporate a number of traits to a transgenic plant besides disease resistance. For example, color, enzyme production, etc. may be desirable traits to confer on a plant. However, transforming plants with a plurality of such traits encounter the same difficulties discussed above with respect to disease resistance. The present invention may be likewise useful in alleviating these problems with respect to traits other than disease resistance.
One problem with transforming plants to contain multiple traits is the possibility that not all of them will be successfully imparted. For example, where there are 4 new traits to be imparted to a transgenic plant, there is a 10% likelihood that each expression event will occur, making the probability of imparting all traits in a plant produced in accordance with the present invention much higher than in a plant transformed with full length trait genes driven by separate promoters. More particularly, the probability of expressing all 4 traits in the latter is 0.0001 (i.e., 0.1xc3x970.1xc3x970.1xc3x970.1), while the probability in the present invention is 0.1.